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Pancreatic cancer is caused by inherited and acquired mutations in specific cancer-associated genes. The discovery of the most common genetic alterations in pancreatic cancer has not only provided insight into the fundamental pathways driving the progression from a normal cell, to non-invasive precursor lesions, to widely metastatic disease, but recent genetic discoveries have also opened new opportunities for gene-based approaches to early detection, personalized treatment, and molecular classification of pancreatic neoplasms.
While “pancreatic cancer” may seem to be a monolithic disease, it is in fact many diseases ranging from low-grade neoplasms such as the pancreatic neuroendocrine tumor, to highly lethal carcinomas such as invasive ductal adenocarcinoma (Table 1) 1. Adding to this complexity, invasive cancers of the pancreas can arise from several histologically distinct non-invasive precursor lesions, and invasive cancers can give rise to clinically discrete patterns of metastasis. While some of the complexity of pancreatic neoplasia can be appreciated grossly and histologically, much of it has only been revealed through detailed genetic analyses2.
The insight that genetic studies of tumors of the pancreas have provided in the last five years has grown logarithmically with the application of next generation sequencing technologies to histologically and clinically well-defined lesions. The exomes of six different tumor types of the pancreas have been sequenced, and large-scale sequencing of the genomes of additional cancers is underway through the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) programs3-7. These sequencing efforts have fundamentally advanced our understanding of pancreatic neoplasia and the time is now ripe to use this understanding to improve patient care.
In this review we will summarize the results of these sequencing efforts and critically analyze the potential clinical implications of recent genetic discoveries. While this review will focus on mutational changes, it should be noted that parallel advances have been made in DNA modification (methylation) and in gene expression, including the expression of microRNAs8-10.
The first tumor type of the pancreas to be sequenced was invasive ductal adenocarcinoma. In a real tour de force, Jones et al. used PCR amplification and Sanger sequencing to analyze the exomes of twenty-four invasive ductal adenocarcinomas of the pancreas, and the hits from the exome sequencing were validated in another 90 pancreatic cancers4. The 24 cancers were found to have an average of 63 genetic alterations, most of which were point mutations, but which also included less common amplifications and deletions. Four genes, all of which were previously known to be targeted in pancreatic cancer, were found to be commonly mutated. These included an oncogene, KRAS, and three tumor suppressor genes, TP53, p16/CDKN2A, and SMAD4 (Table 2). The other genes mutated in these cancers were mutated in only one or two of the tumors and their role in driving pancreatic neoplasia is therefore less clear. Twelve core signaling pathways, including apoptosis, DNA damage control, KRAS signaling, and transforming growth factor-β signaling, were targeted in more than two-thirds of the cancers (Table 3)4. In this issue, A. Le et al describe how an understanding of the metabolic pathways deranged in pancreatic cancer could lead to novel therapeutic targets11.
This genetic sequencing fundamentally defines the “genetic blueprint” of pancreatic cancer. The next step is to determine the timing of these genetic alterations starting with the germline changes, then proceeding through precancerous lesions, and ending with metastases.
Some patients, particularly those with a strong family history of pancreatic cancer, inherit genetic alterations that predispose them to develop pancreatic cancer12-15. Prior to the whole-exome sequencing of pancreatic cancer, four genes, when mutated in the germline, were known to cause the familial aggregation of pancreatic cancer. These genes included BRCA2, p16/CDKN2A, STK11, and PRSS1 (Table 4) 12-15. These familial pancreatic cancer genes are important because information regarding inherited alterations in these loci provide an opportunity to save lives. For example, germline BRCA2 gene mutations not only increase the risk of pancreatic cancer, but they also increase the risk of breast, ovarian, and prostate cancer16-19. Increased surveillance and in selected cases even prophylactic surgery can reduce mortality from these extra-pancreatic neoplasms. In addition, established cancers with biallelic inactivation of the BRCA2 gene may be particularly sensitive to DNA cross-linking agents and to Poly ADP-ribose polymerase (PARP) inhibitors20-22. The BRCA2, p16/CDKN2A, STK11, and PRSS1 genes, however, account for less than 20% of the observed familial aggregation of pancreatic cancer.
The sequencing of pancreatic cancers described earlier by Jones et al. provided a unique opportunity to discover additional familial pancreatic cancer genes. This is because both tumor and germline DNA were sequenced to identify somatic mutations present in the cancer but not in the germline. When Jones et al. carefully studied the germline sequences of the cancers analyzed by whole-exome sequencing, they discovered a pancreatic cancer with a germline PALB2 gene mutation coupled with a second hit to the PALB2 gene in the cancer23. Three additional germline PALB2 mutations were identified in a validation set of 96 familial pancreatic cancer patients, establishing PALB2 as a familial pancreatic cancer gene 23, 24. This discovery demonstrates that exomic sequencing can be used to discover genes responsible for familial pancreatic cancer. This discovery also has clinical implications as it can be used by cancer genetic counselors to assess breast, ovarian and pancreatic cancer risk in their patients, and it can be used by oncologists to guide the treatment of their patients as pancreatic cancers with PALB2 gene mutations may be more sensitive to DNA cross-linking agents such as mitomycin C 25. Indeed, long-term survival has been reported in a patient with a germline PalB2 gene mutation and metastatic pancreatic cancer who was treated with mitomycin C25.
Based on the success of the discovery of PALB2 using whole exome sequencing, Roberts et al sequenced 38 individuals from 16 families in which there was a strong aggregation of pancreatic cancer 26. In two of the families a deleterious germline ATM gene mutations segregated with the disease, and an analysis of one of the affected carrier’s pancreatic cancer revealed a “second hit” to the ATM gene, that is loss of the wild-type allele. Further analyses of additional families suggested that ~2.5% of familial pancreatic cancer may be associated with inherited (germline) ATM gene mutations. These findings not only explain a small fraction of the familial aggregation of pancreatic cancer, but they may, and this is purely speculation, have therapeutic implications as cancers with biallelic inactivation of ATM should theoretically be more sensitive to radiation therapy.
Small (microscopic) precursor lesions, called pancreatic intraepithelial neoplasia (PanIN), have been recognized histologically for close to a century (Figure 1)27, 28. PanINs are important to recognize and characterize because, although small and difficult to detect clinically, they represent an opportunity cure pancreatic neoplasia before an invasive cancer develops. Genetic analyses of PanIN lesions have demonstrated that they harbor many of the same genetic alterations as are found in invasive pancreatic cancer29. Indeed, M. Kanda et al have shown that virtually all of even the lowest grade PanIN lesions (PanIN-1 lesions) harbor mutations of KRAS, p16/CDKN2A, or BRAF30. These genetic findings help establish the sequence of the earliest genetic events that drive pancreatic neoplasia, they add to the growing body of evidence that PanINs are precursors to invasive ductal adenocarcinoma, and suggest that these genetic changes, or their downstream effects, could one day form the basis of an early detection test for curable preinvasive pancreatic neoplasia.
Intraductal papillary mucinous neoplasms (IPMNs) can also be precursors to invasive pancreatic cancer, and they too represent an opportunity cure pancreatic neoplasia before an invasive cancer develops (Figure 2)27. Although not recognized as a distinct entity until the 1980s, IPMNs are growing in importance as these lesions, by definition, are large enough to be detected on clinical imaging. Indeed, with the increasing use of computerized tomography (CT) scanning, the finding of an IPMN is currently one of the more common indications for pancreatic surgery.
In order to understand the nature of this second precursor to invasive pancreatic cancer, Wu et al. sequenced 169 established cancer-associated genes in the cyst fluids of 19 IPMNs6. They discovered: 1) that most IPMNs harbor GNAS and KRAS gene mutations; 2) that both genes have hot spot mutations, codon 201 of GNAS and codon 12 of KRAS; 3) that GNAS mutations are not found in other types of pancreatic cysts; 4) that KRAS and/or GNAS mutations are present in >95% of IPMNs; and 5) that mutations can be detected in cyst fluid. Taken together, these findings suggest that sequencing of endoscopically sampled of cyst fluid for GNAS and KRAS gene mutations could be used to classify a cyst as an IPMN6. Furthermore, quantification of the KRAS and GNAS alleles in IPMN cyst fluids demonstrated that only a small fraction of the alleles (average <25%) are mutant, suggesting that tests designed to detect losses of heterozygosity (LOH) will not work on most IPMN cyst fluid samples.
Wu et al also studied invasive adenocarcinomas that arose in association with IPMNs and found that in most cases (7 of 8) the GNAS gene mutations present in IPMNs are also present in their associated invasive carcinomas6. Just as finding the same genetic mutations in PanINs and invasive ductal adenocarcinomas helped establish PanIN as a bona fide precursor lesion, so too does the finding of GNAS mutations in IPMNs and their associated invasive carcinomas help establish IPMN as a bona fide precursor lesion.
The end stage of the neoplastic progression of pancreatic neoplasia is obviously the development of metastases. Despite the fact that most patients who die from pancreatic cancer die with metastases, historically little attention has been paid to the genetic changes in metastases. Yachida et al. studied the genetic changes in seven metastatic pancreatic cancers, their paired primaries and other metastases from the same patients, and they were able to define the genetic progression of pancreatic neoplasia from invasive cancer to widespread metastases31. In particular, they found that most of the genetic changes in metastases are also present in the paired primary cancers from the same individuals, and that the genetic heterogeneity of metastases mirrors the heterogeneity of the primary carcinoma31. Further analyses of the timing of the genetic changes in these cancers suggested that it takes more than a decade for the initial mutation in a cell in the pancreas to progress to metastatic pancreatic cancer. This latter finding suggests a broad window of opportunity for the early detection of pancreatic neoplasia.
A separate careful study of 76 patients with pancreatic cancer who underwent a rapid autopsy revealed two distinct patterns of disease at the time of death, with ~70% of patients dying with widespread metastatic disease and ~30% with predominantly localized disease with few metastases32. Of interest, SMAD4 loss was associated with widespread metastases, suggesting an underlying genetic basis for the underlying patterns of disease progression and metastasis32.
Iacobuzio-Donahue and colleagues recently applied mathematical modeling to tumor growth data from resected and autopsied patients with pancreatic cancer and found that at the time of clinical diagnosis many pancreatic cancers are in an “exponential” growth phase 33. This suggests that even small delays in the initiation of therapy can have significant deleterious effects.
Pancreatic neoplasia can therefore be seen as the progressive accumulation of genetic alterations. These may start in the germline with inherited mutations (in genes such as BRCA2, p16/CDKN2A, STK11, PALB2, ATM, and PRSS1) in a subset of patients. Individuals with germline alterations as well as those with sporadic disease will progress to non-invasive precursor lesions with the accumulation of mutations in KRAS, p16/CDKN2A, GNAS (in IPMNs), TP53, and SMAD4, and then on to invasive ductal adenocarcinomas harboring over 60 mutations affecting the canonical pathways previously described (Table 3). Inactivation of SMAD4 may contribute to the development of metastases, but much of the genetic heterogeneity found in metastases is already present in the primary carcinoma (Figure 3). As discussed by V.R. Penchev et al in this issue, the heterogeneity of the genetic changes seen in pancreatic cancer may produce some of the heterogeneity observed in so-called cancer stem cells34.
Pancreatic neuroendocrine tumors (PanNETs) are fully malignant neoplasms, but significantly less aggressive than ductal adenocarcinomas1. PanNETs are epithelial neoplasms characterized by a cellular “organoid” histologic pattern of growth, and are defined by neoplastic cells with significant neuroendocrine differentiation. PanNETs therefore provide a unique opportunity to compare the genetic changes in two histologically and clinically distinct neoplasms of the same organ.
Jiao et al. sequenced the exomes of ten pancreatic neuroendocrine tumors (PanNETs)3. Remarkably, the genes targeted in PanNETs were very different from those mutated in invasive ductal adenocarcinoma of the pancreas. The genes commonly targeted in infiltrating ductal adenocarcinomas, TP53, KRAS, p16/CDKN2A and SMAD4, were either never or only rarely mutated in PanNETs3. Conversely, the genes found to be mutated in PanNETs, DAXX, ATRX, MEN1, and the mTOR pathway genes TSC2, PTEN and PIK3CA, were either never or only rarely mutated in ductal adenocarcinomas (Table 5).
The discovery of DAXX and ATRX mutations in PanNETs identified a new cancer pathway. The proteins coded for by DAXX and ATRX play a role in chromatin remodeling, prompting Heaphy et al. to investigate the status of telomeres in PanNETs35. They found that DAXX and ATRX mutations in PanNETs are associated with the alternative lengthening of telomeres (ALT+) phenotype35. While most cancers overcome the “end replication problem” by reactivating telomerase, PanNETs appear to maintain telomere function with alterations in the ATRX or DAXX genes.
The sequencing of PanNETs also has significant clinical implications. Genes coding for members of the mTOR pathway (TSC2, PIK3CA, PTEN) were found to be mutated in 16% of PanNETs, suggesting a possible “personalized” approach to the treatment of PanNETs3. PanNETs that harbor a mutation in a gene coding for a member of the mTOR pathway would be predicted to respond better to mTOR pathway inhibitors, such a everolimus, than would PanNETs lacking one of these mutations36. If this proves true in clinical trials, then patients whose PanNETs harbor a mutation in an mTOR pathway gene could be prioritized to receive an mTOR pathway inhibitor, while patients whose tumors lack one of these mutations could be spared the toxicities of these agents.
The genetic changes in acinar cell carcinomas have not been as well-characterized as have those of other neoplasms of the pancreas37. Unlike ductal adenocarcinomas, acinar cell carcinomas do not usually harbor mutations in KRAS, p16/CDKN2A or SMAD4.37 A fourth of acinar cell carcinomas do have abnormailities in the genes coding for the adenomatous polyposis (APC)/beta-catenin pathway, and isolated acinar cell carcinomas with mismatch repair defects have been described37-39. Acinar cell carcinomas with biallelic BRCA2 gene mutations have also been reported.40
Based on the success of sequencing the exomes of ductal adenocarcinomas of the pancreas and PanNETs, Wu et al sequenced the exomes of the four most common cystic neoplasms of the pancreas: IPMNs, mucinous cystic neoplasms (MCNs), serous cystadenomas (SCNs), and solid-pseudopapillary neoplasms (SPNs) (Figure 2)5. Remarkably, each tumor type was found to have its own mutational profile (Table 6). IPMNs harbored an average of 27 mutations per tumor, and were characterized by mutations in the GNAS, KRAS, and RNF43 genes (RNF43 codes for a protein with intrinsic E3 ubiquitin ligase activity). MCNs harbored an average of 16 mutations per tumor, and MCNs were characterized by mutations in the KRAS, TP53 and RNF43 genes. SCNs had an average of only 10 mutations per tumor and were characterized by mutations in the VHL gene. Finally, SPNs, even though ~10% metastasize, appear to be “one hit wonders.” They had an average of only 2.9 mutations per tumor, and all harbored CTNNB1 gene (β-catenin) mutations5.
Sequencing of the neoplastic epithelium of IPMNs, MCNs, SPNs and SCNs therefore defined a panel of genes that can be used to classify each cyst type5. This finding suggests that genetic analysis of cyst fluid, perhaps obtained by endoscopic ultrasound, could be used to classify definitively a cyst type. This is important because SCNs are virtually always benign, and patients with asymptomatic SCNs could be spared unnecessary surgery if their SCN could be diagnosed non-invasively.
As noted by Drs. Hidalgo and von Hoff in this issue, these are exciting times41. The sequencing of ductal adenocarcinoma, PanNETs, acinar cell carcinomas, and the four types of cystic neoplasms of the pancreas (IPMN, MCN, SCN and SPN) has a number of significant biological and clinical implications.
First, the germline of individuals with a strong family history of pancreatic cancer can be sequenced and those with a genetic predisposition to the disease can be identified (Figure 4, Table 4). These germline changes are, with the exception of germline PRSS1 gene mutations, associated with an increased risk not only of pancreatic cancer, but cancers of other organs as well. Lives can therefore be saved by screening for these extra-pancreatic neoplasms, and hopefully one day by screening for early curable pancreatic neoplasia.
Second, the identification of mutations in precursor lesions such as PanINs and IPMNs has scientific as well as clinical implications. As demonstrated by the discovery of GNAS gene mutations in IPMNs and then the finding of these same mutations in IPMN-associated invasive carcinomas, neoplasia-specific mutations can be used to trace a neoplastic process forward and backward in time6. One could potentially use these genetic changes to identify the earliest neoplastic lesions in the pancreas; lesions that may even appear histologically normal. Such studies on human tissue could easily be supplemented by studies of genetically engineered mouse models. The genetic mutations in precursor lesions could also potentially form the basis for future gene-specific early detection tests, especially since several of the genes targeted in precursor lesions, such as KRAS and GNAS, have hot spot mutations.
Third, as discussed earlier, the identification of a panel of genetic alterations that can be used to distinguish among the four most common cystic neoplasms of the pancreas has significant clinical implications (Figure 4)5. As many as 3% of Americans harbor a cyst in the pancreas that is detectable by computerized tomography42. Distinguishing harmless cysts from precancerous cysts is clinically problematic, but one could potentially do this simply by sequencing endoscopically obtained cyst fluid (Table 6).
Fourth, the results of whole –exome sequencing of six tumor types of the pancreas (2 solid and four cystic) suggests that a new molecular classification of pancreatic neoplasms will soon be possible. This classification would not be an isolated molecular classification, but instead molecular combined with gross, morphology and immunolabeling2. Such a classification has the potential to define new specific clinically relevant tumor types, leading to improved patient care.
Fifth, the sequencing of established invasive cancers has the potential to identify mutations that are therapeutically targetable (Figure 4). For example, patients with a pancreatic cancer with a BRCA2 or PALB2 mutation could be prioritized to receive a PARP inhibitor or a DNA cross-linking agent20, 25. Individualized therapy, albeit for a small minority of patients with pancreatic cancer, can already be achieved.
Sixth, patients with advanced pancreatic cancer can be stratified based on their cancer’s mutational profile. For example, if the SMAD4 results are validated in larger series, the therapeutic approach to patients with borderline resectable pancreatic cancer could potentially be guided by the SMAD4 status of their tumor32. If loss of SMAD4 is associated with an increased risk of widespread metastases, then patients with borderline resectable cancers with SMAD4 loss may not benefit from aggressive surgery. Conversely, borderline resectable cancers with intact SMAD4 may be prioritized for aggressive local therapies.
Finally, mathematical modeling of metastatic pancreatic cancer has also suggested that some cancers are in an “exponential growth phase” at the time of clinical diagnosis33. If this finding is validated, it would support an increased effort to initiate therapy as soon as possible after a patient is diagnosed.
We anticipate that, in the not too distant future, patients with a family history of cancer will have their germline sequenced and their risk predicted. Those at risk will be screened for curable precursor lesions using gene-based tests. Patients diagnosed with an invasive cancer will be biopsied or resected, and a portion of the tumor will be sent for histology and a portion for whole exome sequencing. Therapy will then be determined by the genetic alterations in the tumor (Figure 4).
It is, however, important to note that we present a very optimistic review of the clinical applications of our knowledge of the genetic changes. Most of what we present represents potential impact, and, as discussed by C. Feig et al in this issue, stromal barriers to drug delivery would have to be overcome even if gene-specific targets can be targeted43. Too little has been done to translate molecular discoveries to actual patient care.
Funding: National Institutes of Health
Grants CA 62924 and NCI R01CA97075
Statement of Conflict of Interest: Under a licensing agreement between Johns Hopkins University and Myriad Genetics, Ralph Hruban and Victor E. Velculescu are entitled to a share of royalty payments received by the University on sales of products related to the PALB2 gene. Victor E. Velculescu is a founder of Personal Genome Diagnostics and Inostics, is member of their Scientific Advisory Boards, and owns Personal Genome Diagnostics and Inostics stock, which is subject to certain restrictions under university policy. The terms of these arrangements are managed by Johns Hopkins University in accordance with its conflict-of-interest policies.